1. Field of the Invention
The present invention relates to an enhanced luminescent assay, particularly immunoassay, and to a diagnostic kit for use in the assay. The luminescent assays with which the present invention is concerned are those depending on a chemiluminescent reaction (a chemical reaction that results in the emission of light). The luminescent emission is generally of sufficient duration to enable the light emitted to be detected or measured, and thereby to allow the detection or quantification of an analyte. The chemiluminescent reaction with which this invention is concerned is that between a 2,3-dihydro-1,4-phthalazinedione (DPD), especially luminol or isoluminol, with an oxidant, especially hydrogen peroxide, and a catalyst, especially peroxidase enzyme, which catalyses the oxidation of the DPD by the oxidant. The oxidation is accompanied by emission of light.
2. Description of the Prior Art
Luminescent assays making use of the above-mentioned peroxidase catalysed oxidation of a DPD can be of several major types, the commonest of which are those wherein horseradish peroxidase is conjugated to a ligand in order to label it and a luminescent reaction is used to detect or quantitate the label. The present invention relates exclusively to assays of a different kind, namely those wherein a chemiluminescent compound is used directly to label ligands such as proteins, hormones, haptens, steroids, nucleic acids, metabolites, antigens and/or antibodies. The chemiluminescent DPD such as luminol or isoluminol is normally conjugated to a ligand. Chemiluminescence can be detected by adding peroxidase and an oxidant to the reacted conjugate.
A review of luminescent assays has been published by T. P. Whitehead et. al., Clinical Chemistry 25, 1531-1546 (1979).
The sensitivity of the peroxidase-catalysed chemiluminescent oxidation of DPDs can be enhanced by including in the reagents an enhancer, namely a 6-hydroxybenzothiazole (European Patent 87959B of NRDC), a phenol selected from a narrowly defined class (European Patent 116454B or U.S. Pat. No. 4,598,044 assigned to NRDC) or an aromatic amine selected from a narrowly defined class (UK Patent Application 2162946A of NRDC or European Patent Publication 219352A (Minnesota Mining & Mfg. Co.).
In European Patent Application Publication 210449A (Molecular Diagnostics Inc.) it is alleged that ammonia and water-soluble organic amines are enhancers of chemiluminescent reactions. The amines specifically mentioned are aliphatic polyamines such as spermire, spermidine and butylenediamine, tertiary alkylamines, pyridine, azoles, thiazines, aryl amines and benzylamine. However, little evidence of enhancement is provided in that patent specification.
Despite these prior disclosures, none of which exemplifies enhancement of an assay using direct labelling of a ligand by a DPD, it has been a problem to find amines which enhance such an assay to a high level of light intensity and provide a significantly greater light intensity when the DPD conjugate is present than when it is absent.